The Molecular Phenylpropanoid Pathway
The phenylpropanoid pathway (shown in FIG. 1) produces an array of secondary metabolites including flavones, anthocyanins, flavonoids, condensed tannins and isoflavonoids (Dixon et al., 1996; 2005). In particular, the condensed tannin (CT) biosynthetic pathway shares its early steps with the anthocyanin pathway before diverging to proanthocyanidin biosynthesis.
Anthocyanidins are precursors of flavan-3-ols (e.g. (−)-epicatechin), which are important building blocks for CTs. These cis-flavan-3-ols are formed from anthocyanidins by anthocyanidin reductase (ANR), which has been cloned from many species including A. thaliana and M. truncatula (Xie et al., 2003; 2004). In A. thaliana (−)-epicatechin is the exclusive CT monomer (Abrahams et al., 2002), but in many other species, including legumes, both (+)- and (−)-flavan-3-ols are polymerized to CTs. The biosynthesis of these alternate (+)-flavan-3-ols (catechins) is catalysed by leucoanthocyanidin reductase (LAR). This enzyme has been cloned and characterized from legumes including the CT-rich legume tree Desmodium uncinatum (Tanner et al., 2003), as well as from other species such as grapes and apples (Pfeiffer et al., 2006). The enzyme catalyses the reduction of leucopelargonidin, leucocyanidin, and leucodelphinidin to afzelechin, catechin, and gallocatechin, respectively. No homologues of LAR have been found in A. thaliana, consistent with the exclusive presence of (−)-epicatechin derived CT building blocks in this plant.
Whereas information on TF regulation of this pathway in Arabidopsis seeds is well defined, TFs that control leaf CT biosynthesis within the tribe of Trifolieae have yet to be identified. An important family of TF proteins, the MYB family, controls a diverse range of functions including the regulation of secondary metabolism such as the anthocyanin and CT pathways in plants. The expression of the MYB TF AtTT2 coordinately turns on or off the late structural genes in Arabidopsis thaliana, ultimately controlling the expression of the CT pathway.
An array of Arabidopsis thaliana transparent testa (TT) mutants (Winkel-Shirley, 2002; Debeaujon et al., 2001) and tannin deficient seed (TDS) mutants (Abrahams et al. 2002; 2003) have been made—all being deficient in CT accumulation in the seed coat. Molecular genetic studies of these mutants has allowed for the identification of a number of structural genes and transcription factors (TFs) that regulate the expression and tissue specificity of both anthocyanin and CT synthesis in A. thaliana (Walker et al., 1999; Nesi et al., 2000; 2002).
Although most of the structural genes within the CT pathway have been identified in a range of legumes, attempts to manipulate CT biosynthesis in leaves by engineering the expression of these individual genes has failed so far. The major reason for this is that not one (or a few) enzyme(s) are rate-limiting, but that activity of virtually all enzymes in a pathway has to be increased to achieve an overall increased flux into specific end-products such as condensed tannins.
Transcription factors (TFs) are regulatory proteins that act as repressors or activators of metabolic pathways. TFs can therefore be used as a powerful tool for the manipulation of entire metabolic pathways in plants. Many MYB TFs are important regulators of the phenylpropanoid pathway including both the anthocyanin and condensed tannin biosynthesis (Debaujon et al, 2003; Davies and Schwinn, 2003). For example, the A. thaliana TT2 (AtTT2) gene encodes an R2R3-MYB TF factor which is solely expressed in the seed coat during early stages of embryogenesis, when condensed tannin biosynthesis occurs (Nesi et al., 2001). TT2 has been shown to regulate the expression of the flavonoid late biosynthetic structural genes TT3 (DFR), TT18, TT12 (MATE protein) and ANR during the biosynthesis and storage of CTs. AtTT2 partially determines the stringent spatial and temporal expression of genes, in combination with two other TFs; namely TT8 (bHLH protein) and TTG1 (WD-40 repeat protein; Baudry et al., 2004).
Other MYB TFs in Vitis vinifera; grape (VvMYBPA1) Birdsfoot trefoil and Brassica napus (BnTT2) that are involved in the regulation of CT biosynthesis have also recently been reported (Wei et al., 2007; Bogs et al., 2007; Yoshida et al., 2008).
The AtTT2 gene has also been shown to share a degree of similarity to the rice (Oryza sativa) OsMYB3, the maize (Zea mays) ZmC1, AmMYBROSEA from Antirrhinum majus and PhMYBAN2 from Petunia hybrida, genes which have been shown to regulate anthocyanin biosynthesis (Stracke et al., 2001; Mehrtens et al., 2005).
Condensed Tannins
Condensed tannins (CTs) also called proanthocyanidins (PAs) are colourless polymers, one of several secondary plant metabolites. CTs are polymers of 2 to 50 (or more) flavonoid units (see compound (I) below) that are joined by carbon-carbon bonds which are not susceptible to being cleaved by hydrolysis. The base flavonoid structure is:

Condensed tannins are located in a range of plant parts, for example; the leaves, stem, flowers, roots, wood products, bark, buds. CTs are generally found in vacuoles or on the surface epidermis of the plant
Condensed Tannins in Forage Plants
Forage plants, such as forage legumes, are beneficial in pasture-based livestock systems because they improve both the intake and quality of the animal diet. Also, their value to the nitrogen (N) economy of pastures and to ruminant production are considerable (Caradus et al., 2000). However, while producing a cost-effective source of feed for grazing ruminants, pasture is often sub-optimal when it comes to meeting the nutritional requirements of both the rumen microflora and the animal itself. Thus the genetic potential of grazing ruminants for meat, wool or milk production is rarely achieved on a forage diet.
New Zealand pastures contain up to 20% white clover, while increasing the levels of white clover in pastures helps address this shortfall, it also exacerbates the incidence of bloat. White clover (Trifolium repens), red clover (Trifolium pratense) and lucerne (Medicago sativa) are well documented causes of bloat, due to the deficiency of plant polyphenolic compounds, such as CT, in these species. Therefore the development of forage cultivars producing higher levels of tannins in plant tissue would be a important development in the farming industry to reduce the incidence of bloat (Burggraaf et al., 2006).
In particular, condensed tannins, if present in sufficient amounts, not only helps eliminate bloat, but also strongly influences plant quality, palatability and nutritive value of forage legumes and can therefore help improve animal performance. The animal health and productivity benefits reported from increased levels of CTs include increased ovulation rates in sheep, increased liveweight gain, wool growth and milk production, changed milk composition and improved anthelmintic effects on gastrointestinal parasites (Rumbaugh, 1985; Marten et al., 1987; Niezen et al., 1993; 1995; Tanner et al., 1994; McKenna, 1994; Douglas et al., 1995; Waghorn et al., 1998; Aerts et al, 1999; McMahon et al., 2000; Molan et al., 2001; Sykes and Coop, 2001).
A higher level of condensed tannin also represents a viable solution to reducing greenhouse gases (methane, nitrous oxide) released into the environment by grazing ruminants (Kingston-Smith and Thomas, 2003). Ruminant livestock produce at least 88% of New Zealand's total methane emissions and are a major contributor of greenhouse gas emissions (Clark, 2001). The principle source of livestock methane is enteric fermentation in the digestive tract of ruminants. Methane production, which represents an energy loss to ruminants of around 3 to 9% of gross energy intake (Blaxter and Clapperton, 1965), can be reduced by as much as 5% by improving forage quality. Forage high in CT has been shown to reduce methane emission from grazing animals (Woodward, et al 2001; Puchala, et al., 2005). Increasing the CT content of pasture plants can therefore contribute directly to reduced levels of methane emission from livestock.
Therefore, the environmental and agronomical benefits that could be derived from triggering the accumulation of even a moderate amount of condensed tannins in forage plants including white clover are of considerable importance in the protection and nutrition of ruminants (Damiani et al., 1999).
Legumes
It is the inventors understanding that the regulation of CT foliar-specific pathway in Trifolium legumes, involving the interaction of regulatory transcription factors (TFs) with the pathway, remains unknown. Modification or manipulation of this pathway to influence the amount CT has been explored but, as the process is not straightforward, there has been little firm success in understanding this pathway.
The clover genus, Trifolium, for example, is one of the largest genera in the family Leguminosae (D Fabaceae), with ca. 255 species (Ellison et al., 2006). Only two Trifolium species; T. affine (also known as Trifolium preslianum Boiss. Is) and T. arvense (also known as hare-foot clover) are known to accumulate high levels of foliar CTs (Fay and Dale, 1993). Although significant levels of CTs are present in white clover flower heads (Jones et al., 1976), only trace amounts can be detected in leaf trichomes (Woodfield et al., 1998). Several approaches including gene pool screening and random mutagenesis have failed to provide white or red clover plants with increased levels of foliar CTs (Woodfield et al., 1998).
Genetic Manipulation of Condensed Tannins
The inventors in relation to US2006/012508 created a transgenic alfalfa plant using the TT2 MYB regulatory gene and managed to surprisingly produce CTs constitutively throughout the root tissues. However, importantly, the inventors were unable to achieve CT accumulation in the leaves of this forage legume. It has been previously reported no known circumstances exist that can induce proanthocyanidins (CTs) in alfalfa forage (Ray et al., 2003). The authors of this paper assessed amongst other things whether the LC myc-like regulatory gene (TF) from maize or the C1 myb regulatory gene (TF) from maize could stimulate the flavonoid pathway in alfalfa forage and seed coat. The authors of this paper found that only the LC gene, and not C1 could stimulate anthocyanin and proanthocyanidin biosynthesis in alfalfa forage, but stimulation only occurred in the presence of an unknown stress-responsive alfalfa factor.
Studies assessing condensed tannin production in Lotus plants using a maize bHLH regulatory gene (TF) found that transformation of this TF into Lotus plants resulted in CT's only a very small (1%) increase in levels of condensed tannins in leaves (Robbins et al., 2003).
Previous attempts to alter and enhance agriculturally important compounds in white clover involved altering anthocyanin biosynthesis-derived from the phenylpropanoid pathway. Despite attempts to activate this pathway using several heterologous myc and MYB TFs only one success has been reported, using the maize myc TF B-Peru (de Majnik et al., 2000). All other TFs investigated resulted in poor or no regenerants, implying a deleterious effect from their over-expression.
More recently, TT2 homologs derived from the high-CT legume, Lotus japonicus, have been reported (Yoshida et al., 2008). Bombardment of these genes into A. thaliana leaf cells has shown transient expression resulting in detectable expression of ANR and limited CT accumulation as detected by DMACA. However, these genes have not been transformed and analysed in any legume species.
The expression of the maize Lc gene resulted in the accumulation of PA-like compounds in alfalfa only if the plants were under abiotic stress (Ray et al., 2003). The co-expression of three transcription factors, TT2, PAP1 and Lc in Arabidopsis was required to overcome cell-type-specific expression of PAs, but this constitutive accumulation of PAs was accompanied by death of the plants (Sharma and Dixon, 2005).
Introduction of PAs into plants by combined expression of a MYB family transcription factor and anthocyanidin reductase for conversion of anthocyanidin into (epi)-flavan-3-ol has been attempted by Xie et al. (2006).
This attempt to increase the levels of proanthocyanidins (PAs) in the leaves of tobacco by co-expressing PAP1 (a MYB TF) and ANR were reported as having levels of PAs in tobacco that if translated to alfalfa may potentially provide bloat protection (Xie et al., 2006). Anthocyanin-containing leaves of transgenic M. truncatula constitutively expressing MtANR contained up to three times more PAs than those of wild-type plants at the same stage of development, and these compounds were of a specific subset of PA oligomers. Additionally, these levels of PA produced in M. truncatula fell well short of those necessary for an improved agronomic benefit. The authors state that it remained unclear which additional biosynthetic and non-biosynthetic genes will be needed for engineering of PAs in any specific plant tissue that does naturally accumulate the compounds.
Similar difficulties in expressing CTs or PAs in leaves were also encountered when the TT2 and/or BAN genes were transformed into alfalfa—refer US 2004/0093632 and US 2006/0123508.
Condensed Tannins Useful in Natural Health Products
The use of any flavonoid including proanthocyanidins to form food supplements, compositions or medicaments is also widely known. For example;                US patent application NO: 2003/0180406 describes a method using polyphenol compositions specifically derived from cocoa to improve cognitive function.        Patent publication WO 2005/044291 describes use of grape seed (Vitus genus) to prevent degenerative brain diseases including; stroke, cerebral concussion, Huntington's disease, CJD, Alzheimer's, Parkinsons, and senile dementia.        Patent publication WO 2005/067915 discloses a synergistic combination of flavonoids and hydroxystilbenes (synthetic or from green tea) combined with flavones, flavonoids, proanthocyanidins and anthocyanidins (synthetic or from bark extract) to reduce neuronal degeneration associated with disease states such as dementia, Alzheimer's, cerebrovascular disease, age-related cognitive impairment and depression.        U.S. Pat. No. 5,719,178 describes use of proanthocyanidin extract to treat ADHD.        PCT publication number 06/126895 describes a composition containing bark extract from the genus Pinus to improve, or prevent a decline in, human cognitive abilities or improve, or prevent symptoms of, neurological disorders in a human.        
None of the above considers use of legumes as a raw material source of CT.
It would therefore be useful if there could be provided nucleic acid molecules and polypeptides useful in studying the metabolic pathways involved in flavonoids and/or condensed tannin biosynthesis.
It would also be useful if there could be provided nucleic acid molecules and polypeptides which are capable of altering levels of flavonoids and/or condensed tannins in plants or parts thereof.
In particular, it would be useful if there could be provided nucleic acid molecules which can be used to produce flavonoids and/or condensed tannins in plants or parts thereof de novo.
It is therefore one object of the invention to provide a method to increase CT levels in the leaves of forage legume species. The identification of the gene also provides a method to prevent CT accumulation in legume species which produce detrimental high levels of CT in leaves or seeds.
It would also be useful if there could be provided nucleic acid molecules which can be used alone or together with other nucleic acid molecules to produce plants, particularly forages and legumes, with enhanced levels of flavonoids and/or condensed tannins.
It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice.